Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit VIM-2

Name: Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit VIM-2. ..more

Late stage counterscreen results from the probe development effort to identify common IMP-1 and VIM-2 inhibitors: Epi-absorbance-based biochemical dose response assay for inhibitors of TEM-1 metallo-beta-lactamase

Late stage assay provider counterscreen results from the probe development efforts to identify common IMP-1 and VIM-2 inhibitors: IMP1-transformed E. coli growth inhibition dose response assay in the presence of imipenem

Late stage assay provider counterscreen results from the probe development efforts to identify common IMP-1 and VIM-2 inhibitors: VIM-2-transformed E. coli growth inhibition dose response assay in the presence of imipenem

Late stage assay provider results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: VIM-2-transformed E. coli growth inhibition in the presence of imipenem (synergy)

Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): VIM-2-transformed E. coli growth inhibition in the presence of imipenem (synergy)

Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit VIM-2

Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit IMP-1

Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit IMP-1

Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit AmpC

Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit TEM-1

Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): Growth inhibition of clinically relevant VIM-2-transformed Acinetobacter species (YMC07/8/B3323) in the presence of imipenem (synergy)

Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective):IMP-1-transformed E. coli growth inhibition in the presence of imipenem (synergy)

Name: Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit VIM-2.

Description:

The emergence of gram-negative bacteria that exhibit multi-drug resistance, combined with the paucity of new antibiotics, poses a public health challenge (1). The production of bacterial beta-lactamase enzymes, in particular, is a common mechanism of drug resistance (2-4). The beta-lactamases evolved from bacteria with resistance to naturally-occurring beta-lactams or penams (5), agents which inhibit the transpeptidase involved in cell wall biosynthesis (6). Human medicine adapted these agents into synthetic antibiotics such as penicillins, cephalosporins, carbapenems, and monobactams that contain a 2-azetidone ring (5, 7). The metallo-beta-lactamases (MBL) are zinc-dependent class B beta-lactamases that hydrolyze the beta-lactam ring, rendering the antibiotic ineffective (6, 8). Increasingly, nosocomial beta-lactam antibiotic resistance arises in P. aeruginosa, Enterobacteriaceae, and other pathogenic bacteria via gene transfer of B1 MBLs (4, 9), including IMP (active on IMiPenem) (10) and VIM (Verona IMipenemase) (11, 12). For two of these enzymes, VIM-2 and IMP-1, no inhibitors exist for clinical use (6, 9). Thus, the identification of MBL inhibitors would provide useful tools for reducing nosocomial infections and elucidating their mechanism of action (13).

Assay Overview:The purpose of this assay is to determine the VIM-2 IC50 value of powder samples of compounds identifed as nonselective VIM-2 inhibitor probe candidates and selected analogs. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the VIM-2 substrate, and takes advantage of the fluorescent properties of white microtiter plates (13). Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by beta-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate's emission light (13). In this assay, test compounds are incubated with purified VIM-2 enzyme (0.132 nM) and nitrocefin (60 uM) in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit VIM-2 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in triplicate using a dilution series starting at a nominal test concentration of 60 uM.Protocol Summary:Prior to the start of the assay, 2.5 uL of Assay Buffer (50mM HEPES, 50 uM ZnSO4, 0.05% Brij 35, pH 7.1) containing 0.26 nM VIM-2 protein were dispensed into a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, or DMSO alone (0.45% final concentration) were added to the appropriate wells. The plates were then incubated for 15 minutes at 25 C.The assay was started by dispensing 2.5 uL of 120 uM nitrocefin substrate solution (2X) in Assay Buffer into all wells. After 25 minutes of incubation at 25 C, 5.0 uL of 500 mM EDTA were added to each well to stop the reaction. Next, the plates were centrifuged briefly and well fluorescence was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) (excitation = 480 nm, emission = 530 nm). The Optical density (OD) for each well was calculated according to the following equation:OD = -log ( RFU_SampleWell / RFU_BlankWell )Where:RFU_SampleWell is defined as the raw fluorescence value obtained from test compound wellsRFU_BlankWell is defined as the raw fluorescence value obtained from wells containing Assay BufferThe percent inhibition for each compound was calculated as follows:%_Inhibition = 100 * ( 1 - ( Test_Compound - Median_Positive_Control ) / ( Median_Negative_Control - Median_ Positive _Control ) )Where:Test_Compound is defined as wells containing 0.132nM VIM-2 in the presence of test compound,Negative_Control is defined as wells containing 0.132nM VIM-2 in the presence of DMSO,Positive_Control is defined as wells containing DMSO alone.For this VIM-2 assay, the IC50 value for the S996718 control compound was determined to be 6.443e-5 M.For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 60 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 60 uM.PubChem Activity Outcome and Score:Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.List of Reagents:Recombinant VIM-2 (supplied by Assay Provider)Nitrocefin (BD Diagnostic Systems, part 296289)1536-well plates (Greiner SWSN, part 789175)HEPES (Invitrogen, part 15630)Brij 35 (Sigma-Aldrich, part B4184)Zinc Sulfate (Sigma-Aldrich, part 204986)

These assays were performed by the assay provider. These assays may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.